The Prostate

Effects of Hexanic Extract of Serenoa Repens (PermixonW

160 mg) on Inflammation Biomarkers in the Treatment of
Lower Urinary Tract Symptoms Related to Benign
Prostatic Hyperplasia
Alain Latil,1* Marie-Th
erese P
etrissans,1 J
er^
ome Rouquet,1 Gr
egoire Robert,2
and Alexandre de la Taille3†
1
Institut de Recherche Pierre Fabre, Toulouse, France
2
Department of Urology, Bordeaux Pellegrin University Hospital, Bordeaux, France
3
Department of Urology and INSERM U955 Eq07, CHU Henri-Mondor, Cr
eteil, France

BACKGROUND. Chronic prostatic inflammation (CPI) could be a cause of symptomatic or

complicated benign prostatic hyperplasia (BPH). In previous in vitro and in vivo studies,
1
Hexanic Extract of Serenoa repens (HESr) namely Permixon has demonstrated potent
anti-inflammatory properties. With the aim to provide new insight onto HESr anti-inflamma-
tory properties in human we explore its effect on CPI biomarkers in men with lower urinary
tract symptoms (LUTS) related to BPH using a non-invasive method and investigate links
between biomarkers and clinical symptoms.
METHODS. An international, randomized, double-blind, parallel-group, tamsulosin-con-
trolled study was carried out in 206 men with BPH-related LUTS. Patients received oral daily
HESr 320mg or tamsulosin 0.4 mg during 3 months. The first urine stream after digital rectal
examination (DRE) was collected at Day 1 and Day 90 and mRNA was extracted from
prostatic epithelial cells desquaming in the lumen of the glands and seminal plasma fluid
after DRE. mRNA quantification of the 29 most significant published inflammation markers
in BPH and protein detection in urine was performed.
RESULTS. At D90, a decrease in mean gene expression was observed for 65.4% of the
markers detected in the HESr group versus 46.2% in the tamsulosin group. In the 15 most
frequently expressed genes, this difference was higher (80% vs. 33% respectively). Three
proteins (MCP-1/CCL2, IP-10/CXCL10, and MIF) were detected. At D90, a decrease in the
number of patients who expressed MCP-1/CCL2 and IP-10/CXCL10 was observed only in
the HESr group. Moreover, MIF expression was significantly reduced by HESr compared
with tamsulosin (P ¼ 0.007). Finally, in contrast to tamsulosin, the subgroup of patients
treated by HESr and who over expressed MIF at baseline, had a higher response to the
International Prostate Symptom Score (I-PSS) than those who did not over express this
protein (mean I-PSS change: 6.4 vs. 4.5 respectively). As the study is exploratory, results
should be confirmed in a powered clinical study.

†
International coordinator on behalf of study investigators.
Conflicts of interest: Including specific financial interests and relationships and affiliations relevant to the subject matter discussed in the
manuscript are the following: Alain Latil, Marie-Th
erese P
etrissans and J
er^ ome Rouquet are employees of Pierre Fabre. Gr
egoire Robert is
Investigator in the study and Scientific Advisor. Alexandre de la Taille is the International Study Coordinating Investigator.

Correspondence to: Alain Latil, Institut de Recherche Pierre Fabre, Toulouse, France
E-mail: [email protected]
Received 15 June 2015; Accepted 22 July 2015
DOI 10.1002/pros.23059
Published online in Wiley Online Library
(wileyonlinelibrary.com).

ß 2015 Wiley Periodicals, Inc.

2 Latil et al.

CONCLUSIONS. These results showed for the first time at clinical level the anti-inflamma-
tory properties of HESr, already indicated in BPH-related LUTS. Thus, HESr could be of
interest to prevent unfavourable evolution in patients with CPI. Prostate 9999:XX–XX, 2015.
# 2015 Wiley Periodicals, Inc.

KEY WORDS: benign prostatic hyperplasia; chronic prostatic inflammation; hexanic

extract of serenoa repens; lower urinary tract symptoms; permixonW

INTRODUCTION MATERIALS AND METHODS

A growing body of evidence suggests that Study Participants
chronic prostatic inflammation (CPI) leads to symp-
The study complied with the principles of the
tomatic or complicated benign prostatic hyperplasia
Declaration of Helsinki and was approved by the
(BPH) [1,2]. CPI is a very common condition in
independent Ethics Committees of participating
men over the age of 50 [3]: it has been observed in
centres and countries.
a large proportion of patients treated surgically for
Patients were required to understand and sign the
lower urinary tract symptoms (LUTS) due to
informed consent form and understand and fill in
BPH [4] and in the majority of histological BPH
questionnaires.
tissue obtained from autopsy series [5]. Moreover,
CPI has been associated with higher prostate vol-
ume and a more severe International Prostate Inclusion criteria. To be included in the study, men
Symptom Score (I-PSS) [3,4]. were required to be between 45 and 85 years old with
Histologically, CPI is characterised by the pres- BPH- related LUTS for over 12 months, have an I-PSS
ence of large confluent inflammatory nodules in score 12, prostatic volume 30 cm3 determined by
prostatic tissue [3,4]. These nodules release multiple transrectal ultrasound, maximum flow rate (Qmax) 5–
inflammatory mediators that have been shown to 15 ml/s for a voided volume 150–500 ml, and total
stimulate prostatic cell growth [6]. Nodules also Prostate-specific antigen (PSA) 4 ng/ml or 10 ng/
damage the architecture of the gland, resulting in a ml with ratio PSA (free)/PSA (total) 25% or negative
chain reaction that further sustains the inflamma- prostate biopsy. Patients were required to be free of
tory response and promotes prostatic cell growth, anti-androgens and LH-RH analog for at least
prostatic enlargement, and bladder outlet obstruc- 6 months, 5 alpha-reductase inhibitors and plant
tion [7]. extracts for at least 3 months and alpha blockers and
CPI could be a target for medical treatment in alpha/beta blockers for at least 1 month before
patients with BPH-related LUTS. A recent review screening. Patients taking the following oral medica-
of randomised clinical trials suggested favourable tions at screening required a wash-out of 2 weeks:
effects of non-steroidal anti-inflammatory drugs 5-PDE inhibitors for BPH treatment, NSAIDs cortico-
(NSAIDs) [8], but their side effects related with long-- steroids, or antibiotics by systemic route, mepartri-
term use mostly limit their prescription to acute cine, ACE inhibitors, calcium antagonists, beta
worsening of urinary symptoms. Paubert-Braquet blockers, diuretics, sympathomimetics, antihist-
et al. [9] demonstrated in 1997 that Hexanic Extract of amines, antidepressants (anticholinergic), atropine,
1
Serenoa repens (HESr), namely Permixon and antispasmodic drugs, antiparkinsonism drugs, pseu-
already indicated in BPH-related LUTS could antago- doephedrine, chlorpheniramine, or spironolactone (if
nise 5-lipoxygenase metabolites, leading to an anti-in- unstable dose or initiated 6 weeks or less prior to
flammatory effect. This effect has been recently selection). Moreover, these medications were prohib-
confirmed by in vitro and in vivo studies, indicating ited for the duration of the study.
that HESr can modulate the expression of multiple
inflammation-related genes [10–12]. Non-inclusion criteria. Patients were excluded if
The primary objective of the study was to they had a PVR > 200 ml (by suprapubic ultrasound),
assess the effect of HESr on CPI biomarkers in previous urological history including urethral stric-
men suffering from BPH-related LUTS using a ture disease and/or bladder neck disease, active, or
non-invasive method. The secondary objectives recent (<3 months) or recurrent urinary tract infec-
were to assess the clinical efficacy of HESr tion, urinary retention, indication of BPH surgery,
depending on the prostatic inflammation status of stone in bladder, or urethra, acute, or chronic prosta-
the patients. titis, prostate, or bladder cancer, interstitial cystitis,

The Prostate
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Anti-Inflammatory Properties of Permixon 3

active upper tract stone disease causing symptoms, formed using the TaqMan PreAmp Master Mix Kit
surgery of the prostate, bladder neck or pelvic region. (Life Technologies).
In addition, any local and/or systemic inflammation The mRNA expression of the 29 most significant
disorders, orthostatic hypotension, any neurologic or BPH inflammation markers was quantified [9–19].
psychiatric disease/disorder interfering with the Real-time qPCR was performed with TaqMan Gene
detrusor or sphincter muscle, insulin-dependent dia- Expression Assays (Life Technologie) using the fol-
betes mellitus and non-controlled non-insulin-de- lowing probes:
pendent diabetes mellitus, chronic renal insufficiency, ALOX15 Hs00609608_m1, ALOX15B
history of severe hepatic failure or other severe under- Hs00153988_m1, ALOX5 Hs01095330_m1, CAT
lying disease excluded the participation of the patient Hs00156308_m1, CCL5 Hs00174575_m1, HIF1A
in the study. Hs00153153_m1, LTC4S Hs00168529_m1 MIF
Hs00236988_g1, NFKB1 Hs00765730_m1, PTGES2
Hs00228159_m1, PTGES3 Hs04187821_g1, PTGS2
Study Design
Hs00153133_m1, PTPRC Hs04189704_m1, SELP
This Phase IV trial (https://clinicaltrials.gov/ct2/ Hs00927900_m1, STAT3 Hs00374280_m1, IL17A
show/NCT01604811) was conducted as an interna- Hs00174383_m1, ICOS Hs00359999_m1, CCR7
tional, prospective, randomised, double-blind study Hs01013469_m1, IL1B Hs01555410_m1, IL6
in 2 parallel groups. After a 28–42 day wash-out Hs00985639_m1, IL8 Hs00174103_m1, IL15
period, men suffering from BPH-related LUTS were Hs01003716_m1, PLA2G2A Hs00179898_m1, CXCL10
randomly assigned to receive daily HESr 320 mg Hs01124251_g1, CCL2 Hs00234140_m1, CD40LG
(160 mg B.I.D hard capsule) or tamsulosin LP 0.4 mg Hs00163934_m1, CTLA4 Hs03044418_m1, FGF2
capsule and were followed up over 90 days. Four Hs00266645_m1, CXCL6 Hs00605742_g1 and KLK3
visits were planned for each participant: selection Hs02576345_m1
visit, baseline visit (Day 1), first assessment visit (Day All qPCR reactions were performed with a Quant-
TM
30) and end-of-study visit (Day 90). Studio 6 Flex System (Life Technologies, Foster City,
1
CA, USA) and the TaqMan Gene Expression Master
Mix kit (Life Technologies). The thermal cycling
Methods conditions comprised an initial denaturation step at
95°C for 10 min, 40 cycles at 95°C for 15 sec and
Urine sample collection. Urine samples were col- 60°C for 1 min.
lected from the 203 patients treated with HESr Quantification of KLK3 (PSA gene), specific of
(n ¼ 102) or tamsulosin (n ¼ 101). The first urine prostatic cells, was also performed to confirm that the
stream after digital rectal examination (DRE) was results of markers reflected only the expression of
collected at D1, D30, and D90 in preservation tubes these markers in prostatic cells. The expression of
(Norgen Biotek Corp., Canada). each inflammation marker was therefore normalised
to KLK3. In order to have an overview of inflamma-
RNA isolation and PCR amplification. RNA extrac- tion markers expression in prostatic cells, we also
tion and PCR quantification were performed using quantified these markers in the prostate cell line
standard methods. Briefly, 10 ml were centrifuged at LnCaP and used it as calibrator at baseline.
room temperature for 15 min at 1000g. Two millilitre
of the supernatant were removed and stored at Enzyme-linked immunosorbent assays (ELISA).
80°C for subsequent protein analyses. Protein levels were measured in urine supernatants
Total RNA was isolated from the pellet by using with Quantikine ELISA kits according to the manu-
the RNAble reagent and Qiagen RNeasy mini-preps facturer’s recommendations (R&D Systems Europe
according to the manufacturers’ instructions (Eurobio Ltd, Lille, France). Each experiment was repeated at
and Qiagen, Courtaboeuf, France). The quantity and least 3 times.
purity of extracted RNA were assessed with a Nano-
Drop ND 1000 spectrophotometer (Labtech Interna- Efficacy and safety evaluation. The main efficacy
tional, Paris, France). criterion was the expression level on each mRNA
First-strand cDNA synthesis was performed with gene at D90. The secondary efficacy criteria were the
100 ng of total RNA and SuperScript1 VILO reverse
TM
assessment of I-PSS and Quality of life (QoL) at all
transcriptase (Invitrogen) in a final volume of 20 ml. visits, and sexual function (MSF-4), Qmax (uroflow-
Fourteen multiplex preamplification cycles of 2 metry), Post void residual urine volume (PVR) (supra-
pools of TaqMan Gene Expression Assays (n ¼ 16 for pubic ultrasound) and prostate volume (transrectal
each pool including KLK3 reference gene) was per- ultrasound) at D1, D30, and D90.

The Prostate
4 Latil et al.

A link between mRNA markers/proteins and BPH To account for multiple testing on RNA markers, we
clinical symptoms on changes from baseline was applied a Bonferroni correction, which resulted in an
investigated as well as the analysis of protein expres- adjusted a level of 0.0033. P-values larger than a but
sion profile. Safety criteria included adverse events, less than 0.05 were labelled as “nominally significant”.
physical examination and vital signs at each visit. For the secondary efficacy criteria, I-PSS score,
QoL, MSF-4 and Qmax, changes from baseline were
Statistical considerations. In the absence of previous fitted using a covariance analysis model adjusted for
information on the markers effect size, a sample size baseline value and treatment. These were also
of around 200 patients seemed acceptable to reach the described with respect to the changes of markers.
objectives of this exploratory analysis. For the primary
criterion, downregulation and upregulation of gene
expression were considered to occur when at least a RESULTS
two-fold change from baseline was observed. Wil- Baseline Demographics, Disposition, and Disease
coxon rank-sum tests were used to compare both Characteristics
groups. Next, values were dichotomised as
“expressed“ (value>0) and “not detected“. Values at A total of 206 men were randomised at 36 recruit-
D30 and D90 were fitted together using a method ing centres comprising 26 urologists in 4 countries
based on the generalised estimated equations (GEE). (Spain, Portugal, Italy and France). The patient flow is
Finally, the changes from baseline to D90 were illustrated in Figure 1.
categorised into 4 classes and Cochran-Mantel-Haens- Both treatment groups had similar demographics
zel tests based on the rank score were used to and other BPH baseline characteristics (Table I).
compare treatment groups. Protein expression profiles According to the box plots, mRNA gene expression
were analysed using the same description in classes was globally similar in both groups despite the
and the same test. variability observed (data not shown).

Fig. 1. Patient flow chart.

The Prostate
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Anti-Inflammatory Properties of Permixon 5

TABLE I. Evolution of Clinical Symptoms According to Treatment at D90

HESr n ¼ 102 Tamsulosin n ¼ 101

I-PSS: Total score
Baseline mean (SD) 17.7 (4.4) 16.8 (4.5)
Min/Median/Max 8/18.0/28 12/16.0/30
Value D90 mean (SD) LOCF 13.2 (6.0) 10.3 (5.5)
Change D90-baseline LSMean (SE) 4.28 (0.55) 6.56 (0.55)
QoL score
Baseline mean (SD) 3.9 (0.9) 3.8 (0.9)
Value D90 mean (SD) LOCF 3.0 (1.4) 2.5 (1.2)
Change D90-baseline LSMean (SE) 0.87 (0.12) 1.29 (0.12)
MSF4 score
Baseline mean (SD) 7.4 (4.5) 6.9 (4.5)
Value D90 mean (SD) LOCF 7.7 (4.8) 7.7 (4.7)
Change D90-baseline LSMean (SE) 0.36 (0.35) 0.64 (0.35)
Qmax
Baseline mean (SD) 10.88 (2.69) 10.60 (3.03)
Value D90 mean (SD) LOCF 12.53 (5.21) 12.73 (4.42)
Change D90-baseline LSMean (SE) 1.77 (0.46) 2.09 (0.45)
Transrectal prostate volume
Baseline mean (SD) 48.82 (20.80) 46.29 (13.88)
Value D90 mean (SD) OC 47.95 (20.05) 46.73 (16.83)
Change D90-baseline LSMean (SE) 0.99 (1.08) 0.53 (1.05)
Supra-pubic PVR volume (cm3)
Baseline mean (SD) 53.82 (57.07) 42.04 (47.61)
Value D90 mean (SD) OC 64.11 (63.31) 47.41 (51.29)
Change D90-baseline LSMean (SE) 15.22 (5.80) 4.04 (5.84)

Observed case method (OC).

Last Observation Carried Forward method (LOCF).

Adjusted means from the ANCOVA model: Change ¼ Baseline þ Treatment.

Primary Endpoints groups for the fifteenth marker. In addition, for 11/15
markers (73.3%), upregulation was observed in fewer
Out of the 29 genes investigated at mRNA level, 26
patients in HESr group compared with the tamsulosin
were detected at baseline in at least 1 patient; 3 were
group (4/15 markers—26.6%) (Fig. 2). Thus, combin-
not detected (CD40LG, CTLA4 and ICOS). At D90, a
ing higher decrease and lower increase of these
decrease in mean gene expression was observed for
markers resulted in a favourable effect of HESr in
17/26 (65.4%) markers in the HESr group vs. 12/26
73.3% of mRNA markers (including a nominally
(46.2%) in the tamsulosin group (it should be noted
significant difference for HIF1A: P ¼ 0.008 and
that for 7 of them, a decrease was observed in both
PTGES3: P ¼ 0.0038) compared with 26.6% for tamsu-
groups).
losin (Fig. 3).
We then focused on the most frequently expressed
markers; 15 markers (ALOX5, ALOX15B, CAT, CCL2,
HIF1A, IL1b, IL8, MIF, NFKB1, PLA2G2A, PTGES2, Secondary Endpoints
PTGES3, PTGS2, PTPRC, and STAT3) were expressed
at baseline and D90 in at least 30 patients per group, a Protein expression profile in urine. Based on these
subgroup considered sufficient to be analysed. At mRNA results, 10 proteins were selected, comprising
D90, the decrease in the mean gene expression was 5 cellular proteins (ALOX5, ALOX15B, HIF1A, NFKB,
observed in 80% of these markers in the HESr group and PTPRC) and 5 proteins potentially excreted in
versus 33% in the tamsulosin group (data not shown). urine (MCP-1/CCL2, IL1B, IL8, MIF, and IP-10/
Moreover, for 9/15 markers (60%) downregulation CXCL10). Three proteins (MCP-1/CCL2, IP-10/
was observed more frequently in the HESr group CXCL10, and MIF) were detected.
compared with the tamsulosin group (5/15 markers At D90, a decrease was observed in the number of
—33.3%), No difference was observed between the patients who expressed MCP-1/CCL2 and IP-10/

The Prostate
6 Latil et al.

Fig. 2. Evolution of the 15 most frequently expressed inflammation genes (at least in 30 patients per group at baseline and D90). mRNA
expression was quantified by qPCR. Percentage of patients for whom mRNA expression level was downregulated or upregulated between
baseline and end of treament period (D90). Downregulation and upregulation were considered to occur when a change of at least twofold
between baseline and D90 was observed (see Section 3.5: ’Statistical considerations’). Asterix  denoted nominally significant at P < 0.05.

CXCL10 in the HESr group (mean from 54.8% and respectively for upregulation) (Fig. 4). It is interesting
74.0% at baseline to 35.6% and 63.0% at D90 respec- to note that in the HESr group, MCP-1/CCL2 and
tively) in contrast to the tamsulosin group (mean IP-10/CXCL10 were switched off for 27.4% and 20.5%
from 46.5% and 64.8% at baseline to 47.9% and 67.6% of patients respectively compared with 15.5% and
at D90 respectively). Moreover, in the HESr group, 12.7% of patients respectively in the tamsulosin group
MCP-1/CCL2 and IP-10/CXCL10 were downregu- (data not shown).
lated for a higher percentage of patients (37.0% and MIF protein was expressed in all urine samples at
39.7% respectively) and upregulated for a lower D1 and D90 (Table II). A statistical significance was
percentage of patients (20.5% and 34.3%) compared observed at D90 in favour of HESr with a higher
with the tamsulosin group (28.2% and 31% percentage of patients for whom MIF expression was
respectively for downregulation; 25.4% and 43.7% downregulated (42.5%) compared with the tamsulosin

Fig. 3. Cumulative favourable effect by mRNA gene at the end of treatment (D90). For each mRNA gene, the global favourable effect
corresponding to the sum of the delta of patients between treatment groups for downregulation and delta of patients for less upregulation
was calculated. This was followed by a classification by group. Asterix  denoted nominally significant at P < 0.05.

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Anti-Inflammatory Properties of Permixon 7

Fig. 4. Protein downregulation and upregulation at the end of treatment (D90). Downregulation and upregulation were considered to
occur when a change of at least 25% was observed between D90 and baseline. Treatments were compared using a Cochran-Mantel-Haens-
zel test based on the rank score. Asterik  denoted P < 0.05.

group (23.9%), and a lower percentage of patients for for I-PSS over time in both groups are shown in
whom the MIF expression was upregulated (43.8% vs. Figure 5.
66.2% respectively) (P ¼ 0.007) (Fig. 4).
Link between mRNA expression level and clinical
Clinical outcomes. Compliance to the study treat- symptoms. No apparent relationship was identified
ment was very good and similar in both groups between changes in clinical symptoms and changes in
(>95%). At D90, an improvement in I-PSS, QoL, these markers, but, it should be noted the variability
Qmax and prostate volume was observed in both observed within groups and the small number of
groups. Results are summarised in Table I. The curves patients in subgroups).

TABLE II. Number of Patients who Expressed the Proteins at Baseline and D90

HESr n¼102 Tamsulosin n¼101

Protein detected Number of available data 73 71

MCP 1/CCL2 Baseline Not detected 33 (45.2 %) 38 (53.5 %)
Expressed 40 (54.8 %) 33 (46.5 %)
Value at V4 (D90) Not detected 47 (64.4 %) 37 (52.1 %)
Expressed 26 (35.6 %) 34 (47.9 %)
IP 10/CXCL10 Baseline Not detected 19 (26.0 %) 25 (35.2 %)
Expressed 54 (74.0 %) 46 (64.8 %)
Value at V4 (D90) Not detected 27 (37.0 %) 23 (32.4 %)
Expressed 46 (63.0 %) 48 (67.6 %)
MIF Baseline Not detected — —
Expressed 73 (100.0 %) 71 (100.0 %)
Value at V4 (D90) Not detected — —
Expressed 73 (100.0 %) 71 (100.0 %)

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8 Latil et al.

Fig. 5. I-PSS overtime in both treatment groups. I-PSS: Interna-

tional Prostate Symptom Score; D1: baseline, D30: follow-up visit
Day 30; D90: end of treatment visit Day 90.

Protein expression profile and clinical outcomes.

For the 3 proteins detected, we examined at the
subgroup of patients who over expressed the protein
at baseline (protein value >3rd quartile) compared
with the other patients in the same group and
analysed the I-PSS changes between baseline and D90
by treatment group. Fig. 6. I-PSS adjusted mean change from baseline to end of
No difference was observed between subgroups for treatment (D90). In HESr group, response to I-PSS at D90 was
MCP-1/CCL2 and IP-10/CXCL10. evaluated in patients who over expressed MIF protein at baseline
In contrast, for patients treated by HESr and who (>3rd quartile) to those who did not over express this protein.

over expressed MIF at baseline, a higher response to using Ancova model change¼baselineþtreatment.  >Q3 corre-
I-PSS was observed compared with the other patients sponds to the 25% of patients who expressed MIF at the highest
in the same group (mean I-PSS change: 6.4 vs. 4.5 level.
respectively). This improvement was not observed in
the tamsulosin subgroup (mean I-PSS change: 6.5 With respect to treatment discontinuation, 7.8% of
vs. 6.3) (Fig. 6). patients in the HESr group vs. 3.0% in the tamsulosin
group had at least one adverse event (AE) leading to
study drug discontinuation (Table III).
Safety Assessments
Eight serious adverse events (SAE) were reported DISCUSSION
during the study including 4 during treatment admin-
istration. One (a bilateral gynecomastia), declared in To the best of our knowledge, this study is the
the tamsulosin group, was suspected by the investi- largest randomised clinical trial specifically designed
gator to be in relationship with the treatment. The to investigate the anti-inflammatory effects of medical
percentage of patients with at least one adverse event treatments in patients presenting BPH-related LUTS,
(AE) was 29.4% in the HESr group (41 AEs reported) and the only to use a non-invasive method. The study
versus 30.7% in the tamsulosin group (50 AEs design was rigorous and followed the standards of
reported). A total of 10.8% patients in the HESr group quality clinical research: wash-out period, double--
vs. 8.9% in the tamsulosin group had at least one blind protocol and comparison between different
related treatment-emergent AE. The most frequent active treatments. The baseline characteristics of the
(>2% of patients) treatment-emergent AE (preferred study population were in accordance with the
term) were retrograde ejaculation (4% of patients), required selection criteria. It should be noted the high
constipation (3%) and back pain (3%) with tamsulosin mean I-PSS at baseline in both groups (mean
while no adverse event occurred at a frequency of I-PSS¼17.7 in HESr group and 16.8 in tamsulosin
more than 2% with HESr. No related treatment-emer- group) which corresponds to moderate to severe
gent AE had a frequency over 1% in the HESr group LUTS.
compared with the tamsulosin group in which ejacu- With regard to the lack of placebo arm, given that
lation failure (2%), retrograde ejaculation (2%) and the results showed the superior anti-inflammatory
asthenia (2%) were reported. activity of HESr over tamsulosin, it was considered

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Anti-Inflammatory Properties of Permixon 9

TABLE III. List of Patients who Withdrew From the Study for Safety Reasons (Reported Terms)

Group Subject-Sex-age Adverse event reported term Severity

HESr M-57 Feeling stuffy nose Mild
Palpitation Moderate
M-69 Rash Moderate
M-74 Dizziness sensation Moderate
Persistant tiredness Moderate
M-73 Abdominal pain Moderate
Dry mouth Moderate
Insomnia Moderate
Nightmare Moderate
M-60 Erectile dysfunction Mild
M-68 Groin testicular. the patient suffered from pubic pain/ache Mild
M-67 Diarrhea Moderate
Joint swelling of both hands Moderate
Hypertension Mild
M-46 Epigastric pain Mild
Tamsulosin M-53 Bilateral gynecomastia Moderate
M-61 Anejaculation Mild
M-62 Weight loss (between v2-w3) Moderate

that any potential effect of a placebo would have been and lower-increased genes, favourable anti-inflamma-
even lower than that of tamsulosin. A placebo arm tory activity of HESr was observed in 73.3% of the
would therefore not have provided any additional genes compared with 26.6% of the genes after tamsu-
value regarding the primary endpoint. losin treatment (Fig. 3). Thus, the trend clearly
With regards clinical outcomes, the improvement favoured the anti-inflammatory activity of HESr
in the I-PSS observed in both groups (4.3 and 6.6) compared with tamsulosin.
was in line with already published data on active The favourable effect of tamsulosin observed in
LUTS/BPH treatments, thereby confirming the reli- some genes and/or patients could be explained by the
ability of the clinical findings. obstruction relief associated with alpha-blocker ther-
Although prostatic biopsies remain the reference apy.
method for investigating CPI [3], some urinary bio- MCP-1/CCL2 and IP-10/CXCL10 mean protein
markers were already successfully proposed to assess expressions were clearly reduced after HESr treat-
CPI in BPH with a reliable link between urine and ment, whereas they were slightly raised after tamsu-
tissue samples [14]. It was therefore decided that the losin treatment (Table II). MIF protein expression was
study participants should not be exposed to any risk expressed in all samples and almost 50% of patients
of complication related to an invasive procedure such receiving HESr experienced a significant reduction in
as prostatitis. In this protocol, inflammatory status MIF (Fig. 6).
was monitored with a non-invasive method, which CCL2 encodes MCP-1 (monocyte chemoattractant
allowed for the collection of prostatic epithelial cells protein-1), a chemotactic protein that plays a critical
desquaming in the lumen of the glands and seminal role in the recruitment and activation of monocytes
plasma fluid after DRE. and macrophages in inflammatory diseases [20].
Considering all detected markers, a higher MCP-1/CCL2 downregulation by HESr is in line with
decrease in mean mRNA expression was observed in a previous in vitro study which demonstrated that
HESr group compared to tamsulosin (65% vs. 46% HESr was able to reduce MCP-1/CCL2 expression in
respectively). epithelial and stromal cell lines [10]; MCP-1/CCL2
Regarding the 15 most frequently expressed genes was previously described as the most elevated protein
at baseline, a nominally significant difference in secreted in the prostatic fluid of large prostatic
favour of HESr was observed for HIF1A (P ¼ 0.008) glands [21]. The stimulation of prostatic epithelial
and PTGES3 (P ¼ 0.038), though none remained sig- cells by MCP-1/CCL2 resulted in increased cell
nificant after Bonferroni correction (P < 0.0033) proliferation, potentially leading to prostatic enlarge-
(Fig. 2). Moreover, combining reduced, switched-off, ment [21].

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10 Latil et al.

CXCL10 encodes IP-10 protein (interferon g indu- patients under HESr with higher baseline MIF protein
cible protein 10), which plays an important role in the expression, compared with 4.5 points I-PSS
trafficking of monocytes and activated T cells. When observed in the other HESr subgroup.
activated CD4þT cells, common prostate-infiltrating These results suggest that HESr could be more
cells in BPH patients [7], were co-cultured with BPH effective in patients with higher MIF expression and
cells, a significant increase in IP-10/CXCL10 was higher CPI.
observed [22].
Human prostate stromal fibroblastic cells can secrete
CONCLUSIONS
MCP-1/CCL2 and IP-10/CXCL10 cytokines [13,23]
that are able to recruit and activate CD4þT cells into In this double-blind clinical study, HESr showed
the inflamed prostate, thereby generating an immune for the first time the anti-inflammatory activity in
response leading to the development of chronic immu- men with BPH-related LUTS. HESr, already well--
ne-mediated tissue destruction and fibromyomatosus known as a safe product indicated in the management
growth, as observed in the pathogenesis of BPH [13]. of symptomatic BPH patients, could be particularly
Downregulation of MCP-1/CCL2 and IP-10/CXCL10 useful as an early treatment to prevent unfavourable
by HESr in patients with high CPI could therefore evolution in patients with CPI.
prevent LUTS/BPH progression.
MIF is a long-known T cell cytokine that has been
ACKNOWLEDGMENTS
recognised to be a key mediator of innate immunity
and pleiotropic inflammatory cytokine [24]. MIF has a The authors thank all the investigators involved in
direct chemokine-like function, promotes “directed” the study: Arcangelo Pagliarulo, Rocco Damiano,
cell migration (i.e. chemotaxis) and plays a prominent Matthieu Durand, Emanuele Belgrano, Antonio
role in inflammatory and atherogenic leukocyte Alcaraz, Hakim Fassi-Fehri, Bernard Malavaud, Phil-
recruitment [25]. Another physiological function of ippe Igigabel, Xavier Durand, Raul Martos Calvo,
MIF is to counter-regulate glucocorticoid suppression Luis Campos Pinheiro, Cesare Selli, Riccardo Barto-
of immune cell responses [26]. letti, Henri Chaussade, Jean-Paul Boyes, Richard
MIF plays a pivotal role in the pathogenesis of Yvon, Miguel Unda, Aur
elien Descazeaud, Giorgio
acute and chronic inflammatory diseases by promot- Carmignani, R
egis Soulie, Jacques Tondut, Juan
ing and amplifying involved inflammatory reactions Morote, Alain Palomba, Massimo Porena, Philippe
such as monocyte/macrophage survival or inflamma- Remaud, Francesco Montorsi, Jean-FranSc ois Foucault,
tory cytokine release. Therefore, a direct action of Rafael Medina, Alfredo Rodriguez Antolin, Avelino
HESr on MIF expression makes it an additional Fraga, Jos
e Palma Dos Reis, Francisco Gomez Veiga,
benefit in the management of BPH. However, MIF Beno^ıt Daguzan, G
erard Tatareau, Bernard Boillot,
expression may locally result in higher inflammatory Francisco Javier Burgos, Joaquin Carballido, Jos
e
microenvironment [27], suggesting that the decrease Manuel Cozar, Dario Garcia Rojo, Gilles Karsenty. The
in MIF expression could be the consequence of the authors thank Pierre Bunouf for fruitfull collaboration
overall anti-inflammatory effect of HESr treatment; in in statistical analyses.
this way, we observed that HIF1A is downregulated
under HESr treatment and it has been proved that
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